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Using N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) to

Assay Cyclooxygenase Activity In Vitro

Article in Methods in molecular biology (Clifton, N.J.) · January 2010

DOI: 10.1007/978-1-60761-411-1_9 · Source: PubMed

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 Chapter 9

Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD)

to Assay Cyclooxygenase Activity In Vitro
Nenad Petrovic and Michael Murray

Abstract
Prostaglandin endoperoxide synthase (PGH synthase), also known as cyclooxygenase (COX), was identified
over 30 years ago and is the key enzyme in the pathway by which arachidonic acid is converted to the range
of biologically active lipid mediators known as the prostanoids that participate in numerous physiological
processes. The need for the development of new and improved COX inhibitors as potential therapeutics also
drives the need for rapid, reliable, and inexpensive assays of COX activity. Colorimetric assays are often the
preferred methods of enzyme analysis since they may be readily adapted to simple microplate formats that
require relatively inexpensive and widely available instrumentation. The use of N,N,N ¢,N ¢-tetramethyl-p-
phenylenediamine (TMPD) in high throughput microplate assays of COX activity could become the
approach of choice in the screening of potential therapeutics that inhibit COX activity in vivo. Considering
that TMPD is also a potential substrate for most, if not all, heme peroxidases, it is anticipated that this agent
could find increasing application in the future.

Key words: Enzyme assays, Cyclooxygenases, TMPD, N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine

1. Introduction

1.1. Role of COX COX catalyzes the conversion of arachidonic acid to prostaglan-
in Human Health din (PG) G2 and PGH2. PGH2 is subsequently converted to a
and Disease variety of eicosanoids that include PGE2, PGD2, PGF2a, prostacy-
clin (PGI2), and thromboxane A2 (TXA2). The profile of pros-
tanoids produced within particular cells is dependent on the
enzymes that are expressed in a cell-specific manner. For example,
endothelial cells primarily produce PGI2, whereas platelets mainly
produce TXA2, because these cells express high levels of prostacyclin
synthase and thromboxane synthase, respectively (1).
Two COX isoforms have been identified and are named COX-1
and COX-2 based on the order in which they were ­discovered.

D. Armstrong (ed.), Advanced Protocols in Oxidative Stress II, Methods in Molecular Biology, vol. 594
DOI 10.1007/978-1-60761-411-1_9, © Humana Press, a part of Springer Science + Business Media, LLC 2010

129
130 Petrovic and Murray

Both COX isoenzymes are integral membrane glycoproteins that

are present as homodimers in the membranes of the nucleus and
endoplasmic reticulum. COX-1 and COX-2 have subunit molecu-
lar weights of 70 and 72 kDa. Both COX isozymes have similar
tertiary structures and perform essentially the same catalytic reac-
tion (2). Under normal conditions most tissues express COX-1,
with very low to undetectable levels of COX-2, but the COX-2
isoform is inducible by stress- and growth-related stimuli. The
prostanoids produced by COX-2 have generally been shown to
have pro-inflammatory roles. In contrast, most of the homeostatic
functions of prostanoids are mediated by the constitutively expressed
COX-1. COX-1 and COX-2 also differ in their mRNA stability and
in the preferential utilization of arachidonic acid substrate pools
in tissues (3–5).
The COX isoenzymes and their eicosanoid products play
important functional roles in many physiological processes. In
humans, prostaglandins are involved in diverse functions ranging
from the inflammatory response and blood clotting to ovulation
and initiation of labor. In spite of the fact that drugs such as aspi-
rin have been used widely for over a century, and are now known
to inhibit COX activity, the role of the COX in normal and dis-
ease physiology has only become clearer relatively recently. In
1971 Vane published his seminal observations indicating that the
ability of nonsteroidal antiinflammatory drugs (NSAIDs) to sup-
press inflammation is probably due to their inhibitory actions on
COX enzymes (6). Such inhibition suppresses production of
proinflammatory PGs at the site of injury. Following this discov-
ery, scientists and clinicians have used NSAIDs to dissect the criti-
cal role of the COX enzymes, and the eicosanoids generated by
these pathways, in normal physiology and disease states.
Due to the documented importance of effective COX inhibi-
tion in treating numerous pathological conditions, the search for
new and improved COX inhibitors with greater specificity is
ongoing. Consequently, design of more convenient screening
assays for COX activity is of great importance.

1.2. The Cox Reaction Arachidonic acid, the most important physiological precursor of
eicosanoid biosynthesis, is stored in membrane phospholipids in
an esterified form and can be released intracellularly by the hydro-
lytic action of phospholipases A2 (PLA2) and/or phospholipase C
(PLC). Within the cell arachidonic acid is converted by a range of
enzymes, including COX, to biologically active eicosanoid deriva-
tives. COX actually catalyzes two sequential enzymatic reactions
the bis-oxygenation of arachidonic acid to prostaglandin G2
(PGG2; the cyclooxygenase reaction) and the reduction of PGG2
to prostaglandin H2 (PGH2; the peroxidase reaction). Thus, COX
catalyzes the addition of molecular oxygen at carbon 11 of
­arachidonic acid to form a peroxy intermediate, followed by

Fig. 9.1. The action of COX on arachidonic acid and assays that have measured reaction pathways and their products.
1.3. Methods Used
in the Detection
of COX Activity
Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) 131
r­ earrangement to the cyclic endoperoxide and introduction of a
second molecule of oxygen at carbon 15 to form prostaglandin
G2 (PGG2). Conversion of the endoperoxide PGG2 to the corre-
sponding alcohol (PGH2) employs glutathione as the reducing
cofactor. PGH2 may then be further metabolized to a range of
eicosanoid derivatives (prostaglandins, prostacyclins and throm-
boxanes) by the appropriate synthase (Fig. 9.1).
Various methods for the determination of COX activity have been
developed and are based on the dual enzymatic activities exhibited
by the enzyme: cyclooxygenation and peroxidation. Some of these
assays measure reaction intermediates, whereas others detect the
final products (eicosanoids) of pathways in which COX catalyzes
the rate-limiting step (Fig. 9.1). A range of analytical methodolo-
gies have been applied to the measurement of COX activity. Thus,
oxygen consumption during cyclooxygenation has been measured
using an oxygraph equipped with an oxygen electrode. Various
artificial electron donors have been applied in the measurement of
the peroxidation reaction; these generate colored, fluorescent or
chemiluminescent products. The peroxidation reaction requires a
second substrate which is co-oxidized during hydroperoxide
reduction. COX activity may also be estimated from the turnover
of radioactive arachidonic acid or by measuring its conversion to
labeled PGs (Fig. 9.1). Typically, in vitro assays of COX activity
contain the most efficient substrate, arachidonic acid, an electron
132 Petrovic and Murray

Table 9.1
Methods used to measure COX activity

COX reaction Detection method References

Cyclooxygenation Oxygen consumption (7)

Peroxidation 5-Phenyl-4-pentenyl hydroperoxide (8)

(PPHP) reduction (colorimetry)
N,N,N ¢,N ¢-tetramethyl-p-phenylenedi- (9)
amine (TMPD) oxidation (colorimetry)
10-Acetyl-3,7-dihydroxyphenoxazine (10)
oxidation (fluorometry)
Luminol oxidation (luminescence) (11)

Cyclooxygenation and peroxidation Chromatography of radioactively labelled (12)

products

Prostaglandin synthesis Enzyme-linked immunoassay or (13–15)

radioimmunoassay

donor molecule (usually a low concentration of phenol, about

1 mM, or more specialized electron donors) and hematin. COX
contains Fe3+-protoporphyrin IX as a cofactor which may dissoci-
ate from the protein during its purification, resulting in a mixture
of apo- and holo-enzymes. Therefore, hematin is added to the
reaction mixture (usually at a concentration of 1 mM) in order to
reconstitute maximal enzyme activity. The principal methods for
the assay of COX activity are listed in Table 9.1.

1.4. Direct The most widely used approach for the estimation of total COX
Measurement activity in live cells and tissues is the quantification of prostanoids.
of PG Production Since the COX reaction is considered to be the rate-limiting factor
in prostanoid synthesis (16–18), measurement of PGE2 formation
(the major prostanoid synthesized by the pathway) gives a relative
indication of intracellular COX activity. Enzyme immunoassays
(EIA) are based on the competition of binding of specific tracer
prostanoids, including prostanoid–acetylcholinesterase conjugates,
and those in test samples with antiprostanoid antibodies.
Radioimmunoassays (RIA) measure the binding of antiprostanoid
antibodies to radioactively labeled prostanoids (tracer) and pros-
tanoids in test sample. The quantity of tracer molecules bound to
the antibodies is inversely proportional to the concentration of
competing (unlabeled) prostanoids in the sample. The specific
interaction is then detected either by measuring enzyme activity in
antibody complexes (EIAs), or radioactivity in RIAs (19, 20).

1.5. Ex Vivo Individual COX activity may also be determined in patients by

Prostaglandin use of the ex vivo whole blood assay (15). In these assays, PGE2
Measurements or thromboxane B2 levels in the whole blood (challenged with
 Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) 133

lipopolysaccharide in vitro) are measured by EIA. This approach

can be used to assess the efficacy of selective COX-2 inhibitors in
clinical trials.
These assays detect prostanoids in intact cells or tissues and
are not intended to characterize the COX enzymes. More specific
systems for the determination of COX activity in vitro have
employed RIAs to measure PGE2 formation in microsomal mem-
brane protein preparations containing both COX and PGE2 syn-
thase (21). Several PGE2 synthases have been identified, with the
microsomal PGE2 synthase-1 emerging as a key enzyme in the
formation of PGE2. This assay is relatively complex and is depen-
dent on the quality of the microsomal preparations, because this
influences the PGE2 synthase-1 activity of the sample.

1.6. Detecting An early approach for the direct measurement of COX activity
Radiolabeled in vitro involved the biotransformation of [1-14C] labeled arachi-
COX-Derived Products donic acid. After the reaction, unmetabolized arachidonic acid
of Arachidonic Acid was separated from the products by column chromatography on
silica gel 60. The COX products were then eluted with organic
solvents and the radioactivity in the samples was quantified (12).
Adaptation of high-performance liquid chromatographic meth-
ods, often in conjunction with combinations of mobile phases
and gradient elution, has enabled the resolution of complex mix-
tures of PGs and other eicosanoids.

1.7. Oxygen Oxygen consumption during the first steps of the COX reaction
Consumption may be monitored using an oxygraph equipped with an oxygen
electrode. The reaction is initiated with arachidonate and the ini-
tial rate of oxygen consumption is charted on graph paper (7);
the initial rate of oxygen consumption decreases during the reac-
tion because of oxygen incorporation into arachidonic acid. This
assay is continuous and has the advantage that it directly reflects
COX activity, but is not suitable for high-throughput assays,
requires complex instrumentation and uses relatively high
amounts of enzyme.
Most of the currently used methods for the measurement of
COX activity detect the enzyme’s peroxidase activity in which
PGG2 is reduced to PGH2.

1.8. Co-oxidation An important property of COX is that a range of suitable sub-

Reactions strates can replace PGG2 in the peroxidation step. Thus, COX-
mediated peroxidation activity may be determined conveniently
by the inclusion of reducing substrates that generate products
that are readily quantified. Reduction of 5-phenyl-4-pentenyl
hydroperoxide (PPHP) generates the corresponding 5-phenyl-
4-pentenyl alcohol (PPA; Fig. 9.2) (8) that may be separated by
reverse-phase C18 HPLC and quantified using its absorbance at
252 nm.
134 Petrovic and Murray

Fig. 9.2. Detection of COX peroxidase activity with PHPP.

Fig. 9.3. Detection of COX peroxidase activity with ADHP.

Fig. 9.4. Detection of COX peroxidase activity with Luminol.

Similarly, the reduction of PGG2 by ADHP (10-acetyl-3,7-

dihydroxyphenoxazine) produces the highly fluorescent com-
pound resorufin. ADHP itself is colorless and non-fluorescent,
but is converted during co-oxidation to the red colored, highly
fluorescent product resorufin (Fig. 9.3). ADHP was initially used
to detect horseradish peroxidase activity (9) and has been adapted
by Cayman Chemical (Ann Arbor MI, USA) for screening COX
activity in biological samples.
Co-oxidation of the chemiluminescent substrate luminol is
also used to detect the peroxidase activity of COX; light emission
is recorded with a luminometer (10) (Fig. 9.4).
The artificial electron donor N,N,N ¢,N ¢-tetramethyl-p-
phenylenediamine (TMPD) undergoes co-oxidation by PGG2 to a
blue product (oxidized TMPD) that has an absorbance maximum
at 590 nm (Fig. 9.5). TMPD is a readily oxidizable compound
that serves as a reducing cosubstrate for heme peroxidases (22).
TMPD undergoes one-electron oxidation by heme peroxidase
with two moles of TMPD oxidized per mole of hydroperoxide
reduced by the peroxidase.
The method has been described previously (9, 23), and
­subsequently modified for small volumes added in a microplate
 Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) 135

Fig. 9.5. Detection of COX peroxidase activity with TMPD.

setup (24). TMPD can be used with both crude (cell lysates/
tissue homogenates) and purified enzyme preparations, although
it should be noted that antioxidants in the sample may interfere
with the assay.
Although this is an indirect method, the oxidation of TMPD has
been shown to accurately reflect the rate of conversion of arachidonic
acid to PGH2. The TMPD assay has been used to evaluate potential
new COX inhibitors in a microplate format (26). Peroxidase-
mediated TMPD co-oxidation has been developed commercially by
Cayman Chemical. Consequently, the effects of detergents on COX
activity (27) and the characterization of COX-2 specific inhibitors
DuP 697 and NS-398 (28), as well as the marketed COX-2 inhibitor
celecoxib (29) have been undertaken using the microplate-adapted
TMPD assay. The use of TMPD to detect peroxidase activity in poly-
acrylamide gels illustrates the further potential of this agent in the
detection of a range of hemoproteins (25).

2. Materials

2.1. Equipment 1. Standard 96-well clear plastic plates. They could be obtained
from a variety of suppliers.
2. Visual range spectrophotometer that could accommodate
96-well plates.
3. Automatic multi-plate mixer (shaker) (optional).

2.2. Reagents 1. N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) – Sigma

Aldrich.
2. Recombinant COX-2 (positive control or the source of enzyme
in substrate analysis) – Cayman Chemical.
3. PUFA – (arachidonic and eicosapentaenoic) – Cayman Chemicals.
136 Petrovic and Murray

4. Complete COX assay kit alternative (including TMPD) –

Cayman Chemicals.
5. Hematin – Sigma Aldrich.
6. Dimethyl sulfoxide (DMSO) – Sigma Aldrich.
7. Tris(hydroxymethyl)aminomethane plus HCl – Sigma Aldrich.

3. Methods

3.1. Performance 1. Stock solutions – TMPD stock solution (20 mM) was made in
of the TMPD Assay dimethyl sulfoxide (DMSO) whereas hematin stock solution
(40 mM) was made in 1 M NaOH. PUFA were purchased
from Cayman Chemical as premade solutions in ethanol (see
Note 1). The COX-2 standard was also purchased from
Cayman (see Note 2).
2. Reaction mixture and conditions – All reagents were mixed and
reactions initiated by the addition of a combination of arachidonic
acid (final concentration 50 µM, see Note 3) and TMPD (final con-
centration 100 µM, see Note 4). By varying substrate concentra-
tions from 0.56 to 36 µM, we have found that 10 mM is sufficient
in this assay (Fig. 9.6). Although very short reaction times (up to
2 min) have been used in reported studies, we have found that
10 min gives reproducibly good results and is technically much
easier to perform (Fig. 9.6). Km values for the COX reaction were
consistent (289.1 ± 9.5 nM) over reaction times between 2 and
20 min (Fig. 9.7). Reaction mixture also incorporates 1 µM hematin,

Fig. 9.6. Dependence of TMPD co-oxidation on arachidonic acid concentration and

reaction time.
 Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) 137

Fig. 9.7. Stability of Km values for COX activity over a range of reaction times.

Tris/HCl buffer (100 mM, pH 8) in a final volume of 0.2 mL (see

Notes 5 and 6).
3. Measuring of the oxidized TMPD absorbance – TMPD oxida-
tion is monitored spectrophotometrically in a 96-well-plate
format at 590 nm (see Notes 7 and 8).
4. Data interpretation – The absorbance (590 nm) in the absence of
PUFA should be subtracted from the absorbance in the presence
of PUFA and the net enzyme activity (expressed as the change in
OD at 590 nm, DA590) is then calculated. The reaction rate can
be determined using the TMPD extinction coefficient (adjusted
for the path length of the solution in the microplate) of
0.00826 mM−1 (9). One unit is defined as the amount of enzyme
that will catalyze the oxidation of 1.0 nmol of TMPD per minute
at 25 C. COX activity in units is calculated as follows:
COX activity = DA590 ÷ 10 min ÷ 0.00826\mu M–1 ÷ 0.01# mL
× 1,000 ÷ 2$ = nmol/min/mL (U/mL)

DA590 is the difference in absorbance between wells containing

complete mixture and control wells without PUFA
$
Two molecules of TMPD are required for the reduction
of PGG2 to PGH2
#
0.01 mL is the enzyme sample volume

4. Results

4.1. Testing COX In addition to the testing of potential COX inhibitors, the TMPD
Substrates with assay may also be used to evaluate alternate COX substrates.
TMPD Assay Although arachidonic acid is the most widely studied COX
­substrate, other types of polyunsaturated fatty acids (PUFA), such
138 Petrovic and Murray

Fig. 9.8. In vitro activity of bovine COX-2 determined using AA, SDA, DHA and EPA as substrates. Enzyme kinetic
­parameters (Vmax and Km) were calculated by non-linear regression: filled circle AA; open circle EPA; filled square SDA;
open square DHA ((30), permission granted by Blood journal).

as w-3 PUFA, are also converted by COX into the corresponding

prostanoids. Our findings suggest that w-6 and w-3 PUFA com-
pete for enzymes involved in PUFA biotransformation including
COX ((30), see Note 9). It is widely believed that PUFA biocon-
version enzymes have a greater affinity for w-3 PUFA so that their
biotransformation is favored when the dietary w-3 PUFA intake
is high. Such an effect could lead to the ‘‘competitive inhibition’’
of w-6 PUFA metabolism by w-3 PUFA. In accord with this pos-
sibility, increased intake of the w-3 PUFA eicosapentaenoic acid
(EPA) in man has been shown to suppress the synthesis of arachi-
donic acid-derived eicosanoids and to increase the formation of
the analogous EPA-derived mediators (31). Our data suggest
that, at least in the case of COX-2, the enzyme apparently has a
lower, rather than higher, affinity for w-3 PUFA (Fig. 9.8). These
findings are in accord with the mechanism proposed by Malkowski
et al. (32) where a low rate of EPA oxygenation by COX-1 was
observed and attributed to the presence of the additional double
bond in the molecule, such that the w-3 PUFA adopted a
“strained” conformation in the COX-1 active site. Using the
TMPD assay we have shown that all three w-3 PUFA that were
examined – EPA, docosahexaenoic acid (DHA) and stearidonic
acid (SDA) – are less efficient substrates than the optimal sub-
strate arachidonic acid (AA) (Fig. 9.8).

5. Notes

1. PUFA are light, oxidants and temperature-sensitive. Make

small aliquots in organic solvents (usually ethanol or dimethyl
sulfoxide), store them at −20 C and use them before their
 Using N,N,N ¢,N ¢-tetramethyl-p-phenylenediamine (TMPD) 139

expiration date. Diluted aliquots should be made fresh and

used within a day. TMPD and hematin stock solutions should
be stored similarly.
2. If the source of COX is tissue or cell extract preparation should
be cleared by centrifugation (15 min at 10,000 × g) to mini-
mize turbidity of the sample.
3. If possible, perform the experiments with PUFA at subdued
light.
4. TMPD undergoes spontaneous oxidation. Include controls
without PUFA and subtract their OD from the values obtained
in sample wells.
5. Add all of the ingredients except the PUFA into the wells first.
6. Initiate reaction by adding PUFA last.
7. Since COX assay is completed relatively quickly, use of multi-
channel-pipettor will assure that all the reactions started at the
approximately same time.
8. Use of automatic multiplate mixer (shaker) is advisable to
assure proper mixing of the sample at the beginning of the
reaction.
9. Summary: COX enzymes participate in the formation of a wide
range of eicosanoid mediators. The separate quantification of
each of these fatty acid derivatives in biological samples is time
consuming and has specific analytical requirements. More con-
venient assays that take advantage of the capacity of COX-
derived peroxy intermediates to co-oxidise artificial reducing
substrates, such as TMPD, are now being developed. By this
approach suitable screening methods for potential COX inhibitors
or alternate PUFA substrates have been identified.

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